COVID-19 spike-ACE2 binding assay for drug and antibody screening

ABSTRACT

The present disclosure an ELISA-based assay that uses a glycosylated polypeptide fragment derived from the SARS-CoV-2 spike protein (Covid-19) receptor binding domain (S1RBD) that has affinity for the extracellular domain of Angiotensin Converting Enzyme 2 (ACE2). The S1RBD polypeptide is generated by expression of an encoding nucleic acid by a human cell expression system resulting in glycosylation of the expressed spike receptor binding domain (S1RBD) protein at least at the N343 N-glycosylation site thereof, and which surprisingly and significantly increases the affinity of the S1RBD for ACE2, provides a significant increase in the sensitivity of the assay compared to other known assays.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 63/125,087 entitled “COVID-19 SPIKE-ANGIOTENSIN-CONVERTINGENZYME 2 (ACE2) BINDING ASSAY” filed on Dec. 14, 2020, the entirety ofwhich is hereby incorporated by reference.

SEQUENCE LISTING

This application contains a sequence listing filed in electronic form asan ASCII.txt file entitled “2208031120_ST25” created on Dec. 22, 2020.The content of the sequence listing is incorporated herein in itsentirety.

TECHNICAL FIELD

The present disclosure is generally related to methods of detectingspecific binding between SARS-CoV-2 spike protein and AngiotensinConverting Enzyme 2 (ACE2). The present disclosure is also generallyrelated to kits for the performance of the methods of the disclosure.

BACKGROUND

The coronavirus disease 2019 (COVID-19) pandemic remains an urgentglobal public health concern, with at least 76 million cases reportedand over 1.6 million deaths worldwide as of December 2020. Althoughseveral vaccines are under clinical trials, the number of infections andfatalities will continue to rise for the foreseeable future resulting ina catastrophic impact on societal health and economic development.Numerous medications have been tested for efficacy against COVID-19,notably Remdesivir®, among others, but few of these therapies havedemonstrated robust efficacy in clinical trials. Therefore, hospitalcare of COVID-19 patients will become commonplace world-wide andtreating complications such as cytokine storm and organ failure insevere cases will necessitate increase investigations into the efficacyof new treatments.

The causative agent of COVID-19, severe acute respiratory syndromecoronavirus 2 (SARS-CoV-2) is a member of the Coronaviridae family ofviruses that are known to cause respiratory, hepatic, enteric, andneurologic diseases in mammals. Before 2002, coronaviruses were knownonly as minor human pathogens, contributing to about 15-25% of commoncolds. However, the emergence of a severe outbreak of SARS in 2002caused by the novel coronavirus SARS-CoV propelled public healthvigilance for diseases caused by corona viruses. To date, there areseven known coronavirus of zoonotic origin that can cause human illness,with the coronaviruses MERS-CoV, SARS-CoV, and SARS-CoV-2 identified asbeing causal of severe acute respiratory syndrome.

The coronavirus disease 2019 (COVID-19) is caused by the SARS-CoV-2virus. A critical step of infection is when the virus enters human hostcells, which is enabled by the interaction between the SARS-CoV-2 Spike(S) protein's receptor binding domain (RBD) on the surface of the viralparticle and the Angiotensin I Converting Enzyme 2 (ACE2) receptor onthe surface of human cells. Thus, the identification of small molecules,antibodies such as virus neutralizing antibodies, or other biologicalmolecules that interfere with the formation of the S-ACE2 complex couldhelp to develop drugs to prevent or treat COVID-19.

Currently, one test that may be used is that of Barnhizer & Faro (U.S.patent Ser. No. 10/844,442). This test is a classic Enzyme-LinkedImmunoglobulin Sandwich Assay wherein a polypeptide fragment derivedfrom a human ACE2 protein is immobilized on microtiter surfaces,contacted with a bacteria-expressed fragment of the SARS-CoV-2 Spike (S)protein's receptor binding domain (S1RBD) and, after removal of unboundS1RBD, the ACE2-S1RBD complex is detected using a labeled anti-S1RBDantibody. However, the sensitivity of this test is low, requiringmicrogram amounts of the primary target to obtain a detectable signal.There is, therefore, an ongoing need for a sensitive assay that does notrequire high amounts of the target polypeptides and is suitable for usein high-flow through identification of possible S1RBD/ACE2 bindinginhibitors.

SUMMARY

One aspect of the disclosure, therefore, encompasses embodiments of amethod of detecting binding between the spike-receptor binding domain(S1RBD) of the SARS-CoV-2 (Covid-19) virus and angiotensin-convertingenzyme 2 (ACE2), the method comprising the steps: (a) contacting aglycosylated polypeptide derived from a spike-receptor binding domain(S1RBD) of the SARS-CoV-2 (Covid-19) virus spike (S) protein with apolypeptide derived from a mammalian ACE2, wherein the S1RBD polypeptideor the ACE2-derived polypeptide is bound to the surfaces of wells of amicrotiter plate, wherein the S1RBD polypeptide is a recombinantglycosylated polypeptide expressed from a mammalian cell expressionsystem; (b) washing the wells of unbound polypeptides; (c) either: (i)when the surface bound polypeptide is the ACE2 polypeptide, contactingthe surface bound ACE2 polypeptide with the glycosylated S1RBDpolypeptide, wherein the S1RBD polypeptide further comprises a tagconjugated thereto; or, (ii) when the surface bound polypeptide is theglycosylated S1RBD polypeptide, contacting the surface bound polypeptidewith the ACE2 polypeptide, and then incubating the wells for a periodthat allows the polypeptide bound to the well surfaces to form a complexwith to the polypeptide delivered thereto; (d) washing the wells ofunbound polypeptides; (e) delivering to the wells from step (c)(i) adetectably labeled anti-tag-specific antibody or delivering to the wellsfrom step (c)(ii) a detectably labeled anti-ACE2-specific antibody; (f)incubating the wells for a period to allow the antibody deliveredthereto to bind to the complex formed in either step (c)(i) or (c)(ii);and (g) detecting the label on an antibody bound to the compleximmobilized on the microtiter plate, thereby detecting binding of theS1RBD to the ACE2.

In some embodiments of this aspect of the invention, the polypeptidebound to wells of a microtiter plate can be an ACE2 polypeptide and iscomplexed in step (c)(i) to S1RBD-tag polypeptide delivered to thewells. This method can further comprise the steps: (g) repeating theassay steps (a)-(f) in the presence of a biological sample suspected ofcomprising SARS-CoV-2 (Covid-19) virus, wherein in step (a) the sampleis added to the wells of the microtiter plate; and (h) measuring thedifference between the signal from the detectable label in the absenceand presence of the sample suspected of comprising SARS-CoV-2 (Covid-19)virus, wherein a reduction in the intensity of the signal generated inthe presence of the compound indicates that the sample comprisesSARS-CoV-2 (Covid-19) virus.

In some embodiments of this aspect of the invention the polypeptidebound to wells of a microtiter plate is a glycosylated S1RBD polypeptideexpressed from a mammalian cell expression system and is complexed instep (c)(ii) to the ACE2 polypeptide delivered to the wells.

In some embodiments of this aspect of the invention the tag conjugatedto the S1RBD polypeptide can be an immunoglobulin G (IgG) Fc region andthe anti-tag-specific antibody can be an anti-IgG Fc-specific antibody.

In some embodiments of this aspect of the invention the S1RBDpolypeptide can comprise the amino acid sequence SEQ ID NO: 1.

In some embodiments of this aspect of the invention the S1RBDpolypeptide can comprise the amino acid sequence SEQ ID NO: 1 and isglycosylated at least at the N343 N-glycosylation site thereof.

In some embodiments of this aspect of the invention the label can behorse radish peroxidase (HRP).

In some embodiments of this aspect of the invention the method canfurther comprise the steps: (g) repeating the assay steps (a)-(f) in thepresence of a compound suspected of being an inhibitor of the binding ofthe S1RBD polypeptide to the ACE2 polypeptide or a biological samplesuspected of containing SARS-CoV-2 (Covid-19) virus or an antibodythereto, wherein in steps (c)(i) and (c)(ii) the compound is added tothe wells of the microtiter plate; and (h) measuring the differencebetween a signal from the detectable label in the absence and presenceof the compound suspected of being an inhibitor of the binding of theS1RBD polypeptide to the ACE2 polypeptide, wherein a reduction in theintensity of the signal generated in the presence of the compoundindicates that the compound is an inhibitor of the S1RBD/ACE2 bindingand the degree of the reduction can indicate the magnitude of theinhibition.

In some embodiments of this aspect of the invention the compoundsuspected of being an inhibitor of the binding of the S1RBD polypeptideto the ACE2 polypeptide can be a small molecule, an antibody, or apeptide.

In some embodiments of this aspect of the invention, the antibody can bea monoclonal antibody or in a biological sample isolated from a patientsuspected of having generated anti-SARS-CoV-2 (Covid-19) virusantibodies.

Another aspect of the disclosure encompasses embodiments of a kitcomprising; at least one microtiter plate comprising a plurality ofwells, wherein said wells are coated with an Angiotensin ConvertingEnzyme 2 (ACE2) extracellular domain-derived polypeptide; a plurality ofvessels, wherein said vessels can contain a wash buffer, an assaydiluent, a purified glycosylated SARS-CoV-2 (Covid-19) spike protein RBDregion (S1RBD)-derived polypeptide, wherein the S1RBD polypeptide isobtained by expression from a mammalian cell, and wherein the S1RBDprotein has an immunoglobulin Fc tag conjugated thereto; a horse radishperoxidase-conjugated anti-immunoglobulin G (IgG) Fc-region antibody, aTMB One-Step Substrate Reagent comprising 3,3′,5,5′-tetramethylbenzidine(TMB) in a buffer; and a reaction stop solution comprising about 0.2Msulfuric acid; and instructions for the use of the kit to assay thebinding of the glycosylated S1RBD polypeptide to a domain of ACE2 in theabsence and presence of a compound or a biological sample suspected ofinhibiting said binding.

In some embodiments of the kit of the disclosure, the kit can compriseat least one microtiter plate comprising a plurality of wells, whereinsaid wells are coated with a glycosylated SARS-CoV-2 (Covid-19) spikeprotein RBD region (S1RBD)-derived polypeptide, wherein the S1RBDpolypeptide is obtained by expression from a mammalian cell, a pluralityof vessels, wherein said vessels can contain a wash buffer, an assaydiluent, a purified extracellular domain of a recombinant ACE2polypeptide, a horse radish peroxidase-conjugated anti-ACE2 antibody, aTMB One-Step Substrate Reagent comprising 3,3′,5,5′-tetramethylbenzidine(TMB) in a buffer, and a reaction stop solution comprising about 0.2Msulfuric acid; and instruction for the use of the kit to assay thebinding of the glycosylated S1RBD polypeptide to a domain of ACE2 in theabsence and presence of a compound or biological sample suspected ofinhibiting said binding.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be more readilyappreciated upon review of the detailed description of its variousembodiments, described below, when taken in conjunction with theaccompanying drawings.

FIG. 1 is a diagram showing the spike protein of the SARS-CoV-2(Covid-19) Spike and the S1 receptor binding domain (RBD) (S1RBD)thereof.

FIG. 2 schematically shows an embodiment of the assay method of thedisclosure wherein Angiotensin Converting Enzyme 2 (ACE2) coats thesurfaces of the wells of a microtiter plate, a mammalian cell-expressedS1RBD polypeptide tagged with an immunoglobulin Fc region tag binds tothe ACE2, and detected with an anti-Fc antibody conjugated to adetectable label and which binds to the tag of the bound S1RBDpolypeptide.

FIG. 3 schematically shows an embodiment of the assay method of thedisclosure wherein mammalian cell-expressed S1RBD polypeptide coats thesurfaces of the wells of a microtiter plate, Angiotensin ConvertingEnzyme 2 (ACE2) binds to the mammalian cell-expressed S1RBD, ananti-ACE2 antibody is bound to the ACE2 and detected with an anti-Fcantibody conjugated to a detectable label.

FIG. 4 shows the curve for binding of ACE2 to increasing amounts ofglycosylated S1RBD polypeptide using the method of the disclosure.

FIG. 5 shows the curve for binding of S1RBD-tag polypeptide toincreasing amounts of ACE2 using the method of the disclosure.

FIG. 6 shows that S1RBD protein glycosylation is essential for bindingto ACE2. The S-ACE2 interaction was assessed using untreated anddeglycosylated SARS-CoV-2 S1RBD and untreated and deglycosylated humanACE2 with the binding assay. Untreated and deglycosylated S1 RBDproteins were coated on a 96-well plate respectively. A series ofconcentrations (0, 20, 40, 60, 80, 100 ng/ml) of untreated ordeglycosylated ACE2 protein were added into the wells and bound ACE2protein was detected using anti-ACE2 antibody and HRP conjugatedsecondary antibody. With untreated S1RBD protein, increasing bound ACE2protein, treated or deglycosylated, was detected with increasing ACE2protein concentrations. With deglycosylated S1RBD protein, no increasein bound ACE2 protein, treated or deglycosylated, was detected with anincrease ACE2 protein concentration

FIG. 7 shows that N-linked S1RBD protein glycosylation is essential forbinding to ACE2. The S-ACE2 interaction was assessed using untreated anddeglycosylated SARS-CoV-2 S1RBD with different deglycosylases. S1RBDprotein was treated using different deglycosylases: PNGase F,O-glycosidase, α2-3,6,8,9 neuraminidase A, β1-4 galactosidase S,β-N-acetylhexosaminidase_(f), and Endo H. Untreated and deglycosylatedS1RBD proteins were coated on a 96-well plate respectively. A series ofconcentrations (0, 20, 40, 60, 80, 100 ng/ml) of ACE2 protein were addedinto the wells and bound ACE2 protein was detected using anti-ACE2antibody and HRP conjugated secondary antibody. PNGase F treated S1RBDprotein showed no binding to ACE2 protein.

FIG. 8 shows that N-linked S1RBD protein glycosylation at the N343position of the S1 domain of the spike protein having the amino acidsequence SEQ ID NO: 1 is essential for binding to ACE2. The S-ACE2interaction was assessed using wildtype and mutant of SARS-CoV-2 S1RBD.Mammalian cell-expressed wildtype and three mutant S1RBD proteins,(N331Q, N343Q and N331Q/N343Q) were applied to the binding assay of thedisclosure. Bound ACE2 protein was detected using anti ACE2 antibody andHRP conjugated secondary antibody.

FIG. 9 shows infectivity analysis of a wild type and three glycosylationmutant pseudoviruses rVSV pseudovirus bearing SARS-COV-2 wild type andthree mutants, N331Q, N343Q, and N331/343Q of the RBD of the spikeprotein S1 region of the spike protein were used to drive entry intoA549 cells or Vero cells. At 24 h post infection, pseudotype entry wasanalyzed by determining luciferase activity in cell lysates, and theluminescence relative ratio to the wild type pseudovirus.

FIGS. 10A and 10B illustrate that mammalian cell-expressed SARS-COV-2S1RBD has a better binding activity to ACE2 than does E. coli-expressedS1RBD in vitro.

FIG. 10A shows the ACE2 binding activity of mammalian and E. coliexpressed proteins of SARS-COV-2: S1RBD, S1 and S2 proteins was detectedusing ACE-2 binding assay. The mammalian cell-expressed S1RBD shows thebest S1RBD-ACE2 binding activity compared to E. coli-expressed S1RBD aswell as S1 and S2. Nucleocapsid protein (N) and HIV p24 (P24) wereserved as controls.

FIG. 10B shows the S1RBD-ACE2 binding curve. The mammaliancell-expressed S1RBD shows the dose dependent binding activity (0,3.125, 6.25, 12.5, 25, 50, and 100 ng/ml) compared to the E.coli-expressed S1RBD.

FIG. 11 illustrates mutations of the putative glycosylation sites,including N to Q mutations at 22 putative glycosylation sites, acombination of two glycosylation site mutations in RBD, and threenaturally occurring variants, N74K, N149H, and T719A, with ablatedglycosylation sites.

FIG. 12 shows the amino acid sequence (SEQ ID NO: 1) of the SARS-CoV-2Spike and the S1 receptor binding domain (RBD) expressed from amammalian cell expression system.

FIG. 13 illustrates the ability of the assay method of the disclosure toidentify small molecule inhibitors of the binding of ACE2 and S1RBD.Zafirlukast and cefoperazone, two potent inhibitors of S1RBD-ACE2binding selected by virtual screening, were tested using the S1RBD-ACE2binding assay (cat #CoV-SACE2-1). Camostat Mesilate, a potent serineprotease inhibitor, and DMSO were served as negative controls. A serialdilution (1800, 600, 200, 67 22, 0 ug/ml) of each compound were mixedwith ACE2 protein, and then added to S1RBD protein coated 96-well plate.Unbound ACE2 was removed with washing, and bound ACE2 was detected by ananti-ACE2 antibody and an HRP-conjugated secondary antibody. Theintensity of the yellow color was then measured at 450 nm, and ratio ofsignals (OD) of sample vs. ACE2 alone control was calculated and shown.

FIG. 14 illustrates, using a pseudovirus luciferase assay, that CompoundA (Zafirlukast) identified using the S1RBD-ACE2 binding assay has ablocking effect on pseudovirus entry into host A549 cells.

FIG. 15 illustrates the ability of the assay method of the disclosure toidentify putative monoclonal antibody inhibitors of the binding of ACE2and S1RBD. Monoclonal antibodies against S1RBD were tested using theS1RBD-ACE2 binding assay (cat #CoV-SACE2-1). A serial dilution (1600,800, 400, 200, 100, 0 ng/ml) of each antibody were mixed with ACE2protein, and then added to S1RBD protein coated 96-well plate. UnboundACE2 was removed with washing, and bound ACE2 was detected by ananti-ACE2 antibody and an HRP-conjugated secondary antibody. Theintensity of the yellow color was then measured at 450 nm, and ratio ofsignals of sample vs. ACE2 alone control was calculated and shown.

FIG. 16 illustrates the ability of the assay method of the disclosure toidentify putative monoclonal antibody inhibitors of the binding of ACE2and S1RBD.

FIG. 17 illustrates, using a pseudovirus luciferase assay, that putativemonoclonal antibodies identified as inhibiting S1RBD/ACE2 binding invitro have a blocking effect on pseudovirus entry into host A549 cells.

FIG. 18 illustrates the ability of the assay method of the disclosure toidentify putative monoclonal antibody inhibitors of the binding of ACE2and S1RBD. Monoclonal (mAb) screening for S1-RBD Covid19 Protein usingS1-RBD polypeptide coating the wells of a microtiter plate.

FIG. 19 illustrates, using a pseudovirus luciferase assay, that putativemonoclonal antibodies identified as inhibiting S1RBD/ACE2 binding invitro have a blocking effect on pseudovirus entry into host A549 cells.Pseudoviruses harboring the SARS-COV-2 spike viral surface glycosylationprotein (S1RBD) were incubated with different antibodies at theconcentration of 2 μg/ml at 37° C. for 1 h and inoculated into A549cells. At 24 h post-infection, pseudotype entry was measured bydetermining luciferase activity in cell lysates, and the luminescencerelative ratio to the “zero” concentration data is shown.

FIG. 20 illustrates the evaluation of the diagnostic performance of theACE2 binding assays of the disclosure in detecting COVID19-specificneutralizing antibody in serum.

DETAILED DESCRIPTION

This disclosure is not limited to particular embodiments described, andas such may, of course, vary. The terminology used herein serves thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present disclosure will belimited only by the appended claims.

Where a range of values is provided, each intervening value, to thetenth of the unit of the lower limit unless the context clearly dictatesotherwise, between the upper and lower limit of that range and any otherstated or intervening value in that stated range, is encompassed withinthe disclosure. The upper and lower limits of these smaller ranges mayindependently be included in the smaller ranges and are also encompassedwithin the disclosure, subject to any specifically excluded limit in thestated range. Where the stated range includes one or both of the limits,ranges excluding either or both of those included limits are alsoincluded in the disclosure.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of medicine, organic chemistry, biochemistry,molecular biology, pharmacology, and the like, which are within theskill of the art. Such techniques are explained fully in the literature.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toperform the methods and use the compositions and compounds disclosed andclaimed herein. Efforts have been made to ensure accuracy with respectto numbers (e.g., amounts, temperature, etc.), but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C., and pressure is at or nearatmospheric. Standard temperature and pressure are defined as 20° C. and1 atmosphere.

Before the embodiments of the present disclosure are described indetail, it is to be understood that, unless otherwise indicated, thepresent disclosure is not limited to particular materials, reagents,reaction materials, manufacturing processes, dimensions, frequencyranges, applications, or the like, as such can vary. It is also to beunderstood that the terminology used herein is for purposes ofdescribing particular embodiments only, and is not intended to belimiting. It is also possible in the present disclosure that steps canbe executed in different sequence, where this is logically possible. Itis also possible that the embodiments of the present disclosure can beapplied to additional embodiments involving measurements beyond theexamples described herein, which are not intended to be limiting. It isfurthermore possible that the embodiments of the present disclosure canbe combined or integrated with other measurement techniques beyond theexamples described herein, which are not intended to be limiting.

It should be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a support” includes a plurality of supports. In thisspecification and in the claims that follow, reference will be made to anumber of terms that shall be defined to have the following meaningsunless a contrary intention is apparent.

Each of the applications and patents cited in this text, as well as eachdocument or reference cited in each of the applications and patents(including during the prosecution of each issued patent; “applicationcited documents”), and each of the PCT and foreign applications orpatents corresponding to and/or claiming priority from any of theseapplications and patents, and each of the documents cited or referencedin each of the application cited documents, are hereby expresslyincorporated herein by reference. Further, documents or references citedin this text, in a Reference List before the claims, or in the textitself; and each of these documents or references (“herein citedreferences”), as well as each document or reference cited in each of theherein-cited references (including any manufacturer's specifications,instructions, etc.) are hereby expressly incorporated herein byreference.

Prior to describing the various embodiments, the following definitionsare provided and should be used unless otherwise indicated.

Abbreviations

ELISA, Enzyme Linked Immunoglobulin Sandwich Assay; TMB,3,3,5,5′-tetramethylbenzidine; SARS-CoV-2, Severe Acute RespiratorySyndrome Coronavirus 2; COVID-19, coronavirus disease 2019; RBD receptorbinding domain.

Definitions

The term “specific binding” as used herein refers to the specificrecognition of one molecule, of two different molecules, compared tosubstantially less recognition of other molecules. Generally, themolecules have areas on their surfaces or in cavities giving rise tospecific recognition between the two molecules. Exemplary of specificbinding are antibody-antigen interactions.

The term “antibody” as used herein refers to an immunoglobulin whichspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of another molecule. Theantibody can be monoclonal, polyclonal, or a recombinant antibody, andcan be prepared by techniques that are well known in the art such asimmunization of a host and collection of sera (polyclonal) or bypreparing continuous hybrid cell lines and collecting the secretedprotein (monoclonal), or by cloning and expressing nucleotide sequences,or mutagenized versions thereof, coding at least for the amino acidsequences required for specific binding of natural antibodies.Antibodies may include a complete immunoglobulin or fragment thereof,which immunoglobulins include the various classes and isotypes, such asIgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM, IgY, etc. Fragmentsthereof may include Fab, Fv and F(ab′)2, Fab′, scFv, and the like. Inaddition, aggregates, polymers, and conjugates of immunoglobulins ortheir fragments can be used where appropriate so long as bindingaffinity for a particular molecule is maintained.

Antibodies may be derived from any source, including, but not limitedto, murine spp., rat, rabbit, chicken, human, or any other origin(including humanized antibodies). Techniques for the generation ofantibodies that can specifically recognize and bind to are known in theart.

The term “antigen” as used herein refers to any entity that binds to anantibody and induces at least one shared conformational epitope on theantibody. Antigens can be proteins, peptides, antibodies, smallmolecules, lipid, carbohydrates, nucleic acid, and allergens. An antigenmay be in its pure form or in a sample in which the antigen is mixedwith other components. In particular, the methods of the presentdisclosure are intended to detect human or animal immunoglobulins thatspecifically recognize and bind to epitopes of the S and/or Npolypeptides of the SARS-CoV-2 virus.

The term “Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)”as used herein refers to is the strain of coronavirus that causescoronavirus disease 2019 (COVID-19), the respiratory illness responsiblefor the COVID-19 pandemic. Colloquially known as simply the coronavirus,it was previously referred to by its provisional name, 2019 novelcoronavirus (2019-nCoV), and has also been called human coronavirus 2019(HCoV-19 or hCoV-19). SARS-CoV-2 is a Baltimore class IV positive-sensesingle-stranded RNA virus that is contagious in humans. It is thesuccessor to SARS-CoV-1, the strain that caused the 2002-2004 SARSoutbreak.

Each SARS-CoV-2 virion is 50-200 nm in diameter. Like othercoronaviruses, SARS-CoV-2 has four structural proteins, known as the S(spike), E (envelope), M (membrane), and N (nucleocapsid) proteins; theN protein holds the RNA genome, and the S, E, and M proteins togethercreate the viral envelope. The spike protein, which has been imaged atthe atomic level is responsible for allowing the virus to attach to andfuse with the membrane of a host cell; specifically, its 51 subunitcatalyzes attachment, the S2 subunit fusion.

SARS-CoV-2 has sufficient affinity to the receptor angiotensinconverting enzyme 2 (ACE2) on human cells to use them as a mechanism ofcell entry. Studies have shown that SARS-CoV-2 has a higher affinity tohuman ACE2 than the original SARS virus strain.

Initial spike protein priming by transmembrane protease, serine 2(TMPRSS2) is essential for entry of SARS-CoV-2. After a SARS-CoV-2virion attaches to a target cell, the cell's protease TMPRSS2 cuts openthe spike protein of the virus, exposing a fusion peptide in the S2subunit, and the host receptor ACE2. After fusion, an endosome formsaround the virion, separating it from the rest of the host cell. Thevirion escapes when the pH of the endosome drops or when cathepsin, ahost cysteine protease, cleaves it. The virion then releases RNA intothe cell and forces the cell to produce and disseminate copies of thevirus, which infect more cells.

The term “biological sample” as used herein can refer to a samplederived from blood, preferably peripheral (or circulating) blood. Ablood sample may be, for example, whole blood, blood obtained by afinger prick or a dried blood sample, plasma, serum, or a solubilizedpreparation of such fluids wherein the cell components have been lysedto release intracellular contents into a buffer or other liquid medium.

The terms “binding” as used herein refers to the non-covalentinteractions of the type between a first polypeptide molecule and asecond polypeptide. The strength, or affinity of binding interactionscan be expressed in terms of the dissociation constant (K_(d)) of theinteraction, wherein a smaller K_(d) represents a greater affinity.Binding properties of selected polypeptides can be quantified usingmethods well known in the art. One such method entails measuring therate of complex formation and dissociation of the two interactingpolypeptides, wherein those rates depend on the concentrations of thecomplex partners, the affinity of the interaction, and on geometricparameters that equally influence the rate in both directions. Thus,both the “on rate constant” (K_(on)) and the “off rate constant”(K_(off)) can be determined by calculation of the concentrations and theactual rates of association and dissociation. The ratio ofK_(off)/K_(on) enables cancellation of all parameters not related toaffinity, and is thus equal to the dissociation constant K_(d).

The term “surface” as used herein refers to a solid support such as thesurface of the bottom of a well of a microtiter plate, which areparticularly useful for in vitro assays. Such solid supports might beporous or nonporous, planar or nonplanar and include, but are notlimited to, glass, cellulose, polyacrylamide, nylon, polystyrene,polyvinyl chloride or polypropylene supports. As another example, thepolypeptides of the invention can usefully be attached to the surface ofa microtiter plate for ELISA.

The term “tag” as used herein refers to a moiety conjugated to amolecule such as a peptide or a polypeptide that it is desirable tolabel but does not necessarily have the label attached. A tag can allowa label or labelled moiety to specifically bind to the tag. A tag may bea small molecule to which the labeled moiety can bind, a larger moleculesuch as a peptide, e.g. a hexa-histidine chain, a polypeptide orcombination of polypeptide chains such as, but not limited to, an Fcregion of an antibody any of which may be selectively bound by asuitably selected labeled moiety such as, but not limited to an anti-Fcregion antibody

By “detectably labeled” is meant that a polypeptide or a fragmentthereof, contains a moiety that is elicits a physical or chemicalresponse, a fluorophore or dye, and which can be observed or detected bythe naked eye or by means of instrumentation such as, withoutlimitation, colorimeters, UV spectrophotometers and the like.

The term “detectable moiety” as used herein refers to a label molecule(isotopic or non-isotopic) which is incorporated indirectly or directlyinto a liposomal nanoparticle according to the disclosure, wherein thelabel molecule facilitates the detection of the nanoparticle in which itis incorporated. Thus, “detectable moiety” is used synonymously with“label molecule”. Label molecules, known to those skilled in the art asbeing useful for detection, include chemiluminescent or fluorescentmolecules. Various fluorescent molecules are known in the art which aresuitable for use to label a nucleic acid for the method of the presentinvention. The protocol for such incorporation may vary depending uponthe fluorescent molecule used. Such protocols are known in the art forthe respective fluorescent molecule.

The term “dye” as used herein refers to any reporter group whosepresence can be detected by its light absorbing or light emittingproperties. For example, Cy5 is a reactive water-soluble fluorescent dyeof the cyanine dye family. Cy5 is fluorescent in the red region (about650 to about 670 nm). It may be synthesized with reactive groups oneither one or both of the nitrogen side chains so that they can bechemically linked to either nucleic acids or protein molecules. Labelingis done for visualization and quantification purposes. Cy5 is excitedmaximally at about 649 nm and emits maximally at about 670 nm, in thefar-red part of the spectrum; quantum yield is 0.28. FW=792. Suitablefluorophores(chromes) for the probes of the disclosure may be selectedfrom, but not intended to be limited to, fluorescein isothiocyanate(FITC, green), cyanine dyes Cy2, Cy3, Cy3.5, Cy5, Cy5.5 Cy7, Cy7.5(ranging from green to near-infrared), Texas Red, and the like.Derivatives of these dyes for use in the embodiments of the disclosuremay be, but are not limited to, Cy dyes (Amersham Bioscience), AlexaFluors (Molecular Probes Inc.), HILYTE™ Fluors (AnaSpec), and DYLITE™Fluors (Pierce, Inc).

The term “fluorophore” as used herein refers to any reporter group whosepresence can be detected by its light emitting properties.

The term “immobilized on a solid support” as used herein refers to apolypeptide attached to a substrate at a particular location so that itmay be subjected to washing or other physical or chemical manipulationwithout being dislodged. A number of solid supports and immobilizingmethods are known in the art, and may be used in the methods of thisdisclosure.

The terms “expressed” and “expression” as used herein refer to thetranscription from a gene to give an RNA nucleic acid molecule at leastcomplementary in part to a region of one of the two nucleic acid strandsof the gene. The term “expressed” or “expression” as used herein alsorefers to the translation from said RNA nucleic acid molecule to give aprotein, an amino acid sequence or a portion thereof.

The term “fragment” of a protein or nucleic acid as used herein refersto any portion of the amino acid sequence.

The term “immunoglobulin” as used herein refers to a class of proteinsthat exhibit antibody activity and bind to other molecules (e.g.,antigens and certain cell-surface receptors) with a high degree ofspecificity. Immunoglobulins can be divided into five classes: IgM, IgG,IgA, IgD, and IgE. IgG is the most abundant antibody class in the bodyand assumes a twisted “Y” shape configuration. With the exception of theIgMs, immunoglobulins are composed of four peptide chains that arelinked by intrachain and interchain disulfide bonds. IgGs are composedof two polypeptide heavy chains (H chains) and two polypeptide lightchains (L chains) that are coupled by non-covalent disulfide bonds.

The light and heavy chains of immunoglobulin molecules are composed ofconstant regions and variable regions. For example, the light chains ofan IgG1 molecule each contain a variable domain (V_(L)) and a constantdomain (C_(L)). The heavy chains each have four domains: an aminoterminal variable domain (V_(H)), followed by three constant domains(C_(H)1, C_(H)2, and the carboxy terminal C_(H)3). A hinge regioncorresponds to a flexible junction between the C_(H)1 and C C_(H)2domains. Papain digestion of an intact IgG molecule results inproteolytic cleavage at the hinge and produces an Fc fragment thatcontains the C_(H)2 and C_(H)3 domains, as well as two identical Fabfragments that each contain a C_(H)1 C_(L), V_(H), and V_(L) domain. TheFc fragment has complement- and tissue-binding activity. The Fabfragments have antigen-binding activity

Immunoglobulin molecules can interact with other polypeptides through acleft within the C_(H)2-C_(H)3 domain. This “C_(H)2-C_(H)3 cleft”typically includes the amino acids at positions 251-255 within theC_(H)2 domain and the amino acids at positions 424-436 within the C_(H)3domain. As used herein, numbering is with respect to an intact IgGmolecule as in Kabat et al. (Sequences of Proteins of ImmunologicalInterest, 5th ed., Public Health Service, U.S. Department of Health andHuman Services, Bethesda, Md.). The corresponding amino acids in otherimmunoglobulin classes can be readily determined by those of ordinaryskill in the art.

The Fc region can bind to a number of effector molecules and otherproteins, including the cellular Fe Receptor that provides a linkbetween the humoral immune response and cell-mediated effector systems(Hamano et al., (2000) J. Immunol. 164: 6113-6119; Coxon et al., (2001)Immunity 14: 693-704; Fossati et al., (2001) Eur. J. Clin. Invest. 31:821-831). The FC□ receptors are specific for IgG molecules, and includeFcγRI, FcγRIIa, FcγRIIb, and FcγRIII. These isotypes bind with differingaffinities to monomeric and immune-complexed IgG.

The term “polypeptide” includes proteins and fragments thereof.Polypeptides are disclosed herein as amino acid residue sequences.

The term “expressed” or “expression” as used herein refers to thetranscription from a gene to give an RNA nucleic acid molecule at leastcomplementary in part to a region of one of the two nucleic acid strandsof the gene. The term “expressed” or “expression” as used herein alsorefers to the translation from said RNA nucleic acid molecule to give aprotein, an amino acid sequence or a portion thereof.

The term “recombinant” as used herein, and referring to a nucleic acidmolecule, means a polynucleotide of genomic, cDNA, semisynthetic, orsynthetic origin which, by virtue of its origin or manipulation: (1) isnot associated with all or a portion of the polynucleotide with which itis associated in nature; and/or (2) is linked to a polynucleotide otherthan that to which it is linked in nature. The term “recombinant” asused with respect to a protein or polypeptide means a polypeptideproduced by expression of a recombinant polynucleotide

The term “engineered protein” as used herein refers to anon-naturally-occurring polypeptide. The term encompasses, for example,a polypeptide that comprises one or more changes, including additions,deletions or substitutions, relative to a naturally occurringpolypeptide, wherein such changes were introduced by recombinant DNAtechniques. The term also encompasses a polypeptide that comprises anamino acid sequence generated by man, an artificial protein, a fusionsprotein, and a chimeric polypeptide. Once expressed, recombinantpeptides, polypeptides and proteins can be purified according tostandard procedures known to one of ordinary skill in the art, includingammonium sulfate precipitation, affinity columns, column chromatography,gel electrophoresis and the like. Substantially pure compositions ofabout 50 to 99% homogeneity are preferred, and 80 to 95% or greaterhomogeneity are most preferred for use as therapeutic agents.

The term “detectable label” as used herein refers to a moiety attachedto a specific binding partner, such as an antibody or an analyte, e.g.,to render the reaction between members of a specific binding pair, suchas an antibody and an analyte, detectable, and the specific bindingpartner, e.g., antibody or analyte, so labeled is referred to as“detectably labeled.” Thus, the term “labeled binding protein” as usedherein, refers to a protein with a label incorporated that provides forthe identification of the binding protein. In an embodiment, the labelis a detectable marker that can produce a signal that is detectable byvisual or instrumental means, e.g., incorporation of a radiolabeledamino acid or attachment to a polypeptide of biotinyl moieties that canbe detected by marked avidin (e.g., streptavidin containing afluorescent marker or enzymatic activity that can be detected by opticalor colorimetric methods). Examples of labels for polypeptides include,but are not limited to, the following: radioisotopes or radionuclides,chromogens, fluorescent labels (e.g., FITC, rhodamine, and lanthanidephosphors), enzymatic labels (e.g., horseradish peroxidase, luciferase,alkaline phosphatase); chemiluminescent markers; biotinyl groups;predetermined polypeptide epitopes recognized by a secondary reporter(e.g., leucine zipper pair sequences, binding sites for secondaryantibodies, metal binding domains, and epitope tags); and magneticagents, such as gadolinium chelates. Representative examples of labelscommonly employed for immunoassays include moieties that produce light,e.g., acridinium compounds, and moieties that produce fluorescence,e.g., fluorescein. Other labels are described herein. In this regard,the moiety itself may not be detectably labeled but may becomedetectable upon reaction with yet another moiety. Use of “detectablylabeled” is intended to encompass the latter type of detectablelabeling.

The term “monoclonal antibody” or “mAb” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigen. Furthermore, in contrast topolyclonal antibody preparations that typically include differentantibodies directed against different determinants (epitopes), each mAbis directed against a single determinant on the antigen. The modifier“monoclonal” is not to be construed as requiring production of theantibody by any particular method.

The term “recombinant” as used herein, and referring to a nucleic acidmolecule, means a polynucleotide of genomic, cDNA, semisynthetic, orsynthetic origin which, by virtue of its origin or manipulation: (1) isnot associated with all or a portion of the polynucleotide with which itis associated in nature; and/or (2) is linked to a polynucleotide otherthan that to which it is linked in nature. The term “recombinant” asused with respect to a protein or polypeptide means a polypeptideproduced by expression of a recombinant polynucleotide

the term “engineered protein” as used herein refers to anon-naturally-occurring polypeptide. The term encompasses, for example,a polypeptide that comprises one or more changes, including additions,deletions or substitutions, relative to a naturally occurringpolypeptide, wherein such changes were introduced by recombinant DNAtechniques. The term also encompasses a polypeptide that comprises anamino acid sequence generated by man, an artificial protein, a fusionsprotein, and a chimeric polypeptide. Once expressed, recombinantpeptides, polypeptides and proteins can be purified according tostandard procedures known to one of ordinary skill in the art, includingammonium sulfate precipitation, affinity columns, column chromatography,gel electrophoresis and the like. Substantially pure compositions ofabout 50 to 99% homogeneity are preferred, and 80 to 95% or greaterhomogeneity are most preferred for use as therapeutic agents.

The term “small molecule” as used herein refers to an organic compound,including an organometallic compound, of a molecular weight less thanabout 2 kDa, that is not a polynucleotide, a polypeptide, apolysaccharide, or a synthetic polymer composed of a plurality ofrepeating units.

The term “polypeptide” as used herein, refers to any polymeric chain ofamino acids. The terms “peptide” and “protein” are used interchangeablywith the term polypeptide and also refer to a polymeric chain of aminoacids. The term “polypeptide” encompasses native or artificial proteins,protein fragments and polypeptide analogs of a protein sequence. Apolypeptide may be monomeric or polymeric. Use of “polypeptide” hereinis intended to encompass polypeptide and fragments and variants(including fragments of variants) thereof, unless otherwise stated.

The term “sample,” as used herein, is used in its broadest sense. A“biological sample,” as used herein, includes, but is not limited to,any quantity of a substance from a living thing or formerly livingthing. Such living things include, but are not limited to, humans, mice,rats, monkeys, dogs, rabbits and other animals. Such substances include,but are not limited to, blood, (e.g., whole blood), plasma, serum,urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes,monocytes, other cells, organs, tissues, bone marrow, lymph nodes andspleen.

The term “control” refers to a composition known to not contain analyte(“negative control”) or to contain analyte (“positive control”). Apositive control can comprise a known concentration of analyte.“Control,” “positive control,” and “calibrator” may be usedinterchangeably herein to refer to a composition comprising a knownconcentration of analyte. A “positive control” can be used to establishassay performance characteristics and is a useful indicator of theintegrity of reagents (e.g., analytes).

The terms “specific” and “specificity” as used herein are in the contextof an interaction between members of a specific binding pair (e.g., anantigen (or fragment thereof) and an antibody (or antigenically reactivefragment thereof)) refer to the selective reactivity of the interaction.The phrase “specifically binds to” and analogous phrases refer to theability of antibodies (or antigenically reactive fragments thereof) tobind specifically to analyte (or a fragment thereof) and not bindspecifically to other entities.

The term “Angiotensin-Converting Enzyme 2 (ACE2)” as used herein is anenzyme attached to the cell membranes of cells located in the lungs,arteries, heart, kidney, and intestines. ACE2 lowers blood pressure bycatalyzing the hydrolysis of angiotensin II (a vasoconstrictor peptide)into angiotensin (1-7) (a vasodilator). ACE2 counters the activity ofthe related angiotensin-converting enzyme (ACE) by reducing the amountof angiotensin-II and increasing Ang(1-7), making it a promising drugtarget for treating cardiovascular diseases. ACE2 serves as the entrypoint into cells for some coronaviruses, including HCoV-NL63, SARS-CoV,and SARS-CoV-2.

Angiotensin-converting enzyme 2 is a zinc-containing metalloenzymelocated on the surface of endothelial and other cells. ACE2 proteincontains an N-terminal peptidase M2 domain and a C-terminal collectrinrenal amino acid transporter domain. ACE2 is a single-pass type Imembrane protein, with its enzymatically active domain exposed on thesurface of cells in the lungs and other tissues. The extracellulardomain of ACE2 is cleaved from the transmembrane domain by anotherenzyme known as sheddase, and the resulting soluble protein is releasedinto the bloodstream and ultimately excreted as urine.

One fragment of the ACE2 advantageous in the methods of the disclosureis isolated from the human AVE2 having the Accession No: Q9BYF1 and isthe expressed Region Gln18-Ser740 (Extracellular domain) (SEQ ID NO: 2).

Phrases such as “under conditions suitable to provide” or “underconditions sufficient to yield” or the like, in the context of methodsof synthesis, as used herein refers to reaction conditions, such astime, temperature, solvent, reactant concentrations, and the like, thatare within ordinary skill for an experimenter to vary, that provide auseful quantity or yield of a reaction product. It is not necessary thatthe desired reaction product be the only reaction product or that thestarting materials be entirely consumed, provided the desired reactionproduct can be isolated or otherwise further used.

The term “lateral flow tests as used herein, also known as lateral flowimmunochromatographic assays, refers to devices intended to detect thepresence of a target substance in a liquid sample without the need forspecialized and costly equipment. These tests are widely used in medicaldiagnostics for home testing, point of care testing, or laboratory use.These tests are simple, economic and generally show results in around5-30 minutes.

Lateral flow tests operate on the same principles as the enzyme-linkedimmunosorbent assays (ELISA). Tests run the liquid sample along thesurface of a pad with reactive molecules that show a visual positive ornegative result. The pads are based on a series of capillary beds, suchas pieces of porous paper, microstructured polymer. The pads have thecapacity to transport fluid (e.g., urine, blood, saliva) spontaneously.

The sample pad acts as a sponge and holds an excess of sample fluid.Once soaked, the fluid flows to the second conjugate pad in which can bestored freeze dried bio-active particles called conjugates (see below)in a salt-sugar matrix. A conjugate pad contains all the reagentsrequired for an optimized reaction between the target molecule (e.g.,ACE2 protein) and its partner (e.g., S1RBD-tag polypeptide) that hasbeen immobilized on the particle's surface. This marks target particlesas they pass through the pad and continue across to the test and controllines. The test line shows a signal. The control line contains affinityligands (in the context of the assays of the disclosure, an anti-tagantibody) that show whether the sample has flowed through and thebio-molecules in the conjugate pad are active. After passing thesereaction zones, the fluid enters the final porous material, the wick,that simply acts as a waste container.

Discussion

The present disclosure encompasses embodiments of a variant ELISA-basedassay that employs a glycosylated polypeptide fragment derived from thereceptor binding domain (S1RBD) of the SARS-CoV-2 (Covid-19) virus spikeprotein and that has affinity for the extracellular domain ofAngiotensin Converting Enzyme 2 (ACE2). The assays of the disclosureemploy the ability of such a fragment to bind to the mammalian cellsurface receptor Angiotensin Converting Enzyme 2 (ACE2) used by theSARS-CoV-2 (Covid-19) virus as a means for entry into a human cell.Accordingly, the assays of the disclosure are in two forms, as shown inFIGS. 2 and 3, one where the spike-derived polypeptide is immobilized ona surface, such as the bottom surface of a well of a microplate known inthe art, and is then contacted by a buffered solution of a polypeptidederived from a mammalian ACE2 protein. In the alternative, it is theACE2-derived polypeptide that is immobilized and contacted with the S1RBD spike polypeptide fragment.

A notable feature of the S protein of SARS-CoV-2 is that it isextensively decorated with up to a hundred N-linked glycans, a processthat occurs by viral hijacking of the host's glycosylation pathways.Glycosylation of viral structures such as S proteins, contributes to theviruses host immune evasion strategies through the masking antigenicepitopes. Structural data along with glycoproteomics analyses haveproposed that extensive glycosylation of the spike protein shieldsagainst neutralizing antibodies access (Xiong et al., (2018) J. Virol.92(4): 1-16). Importantly, the glycans on S protein possibly have anunappreciated role in both the stability of S and resultant host cellreceptor interactions and cell membrane fusion during entry into thehost cell. This gap in the knowledge underscores an exigent need forcharacterizing the relative influence of SARS-CoV-2 S proteinglycosylation in identifying the molecular basis of tis interaction withACE2, and the influence of glycans on infectivity.

It has now been found that N-glycosylation of SARS-CoV-2 is necessaryfor in vitro binding to ACE2 and for the entry of pseudovirus intocells. The glycosylated residues Asn343 and Asn331 on S1 RBD play a keyrole in its binding to ACE2 as well as infectivity. Together, these datasupport the conclusion that N-glycosylation of Asn343 and Asn331 iscrucial for the S-ACE2 interaction and infectivity. These data allowedfor the development of the assays of the disclosure.

Unlike with other ELISA-based assays known in the art, the spike S1RBDpolypeptide is generated by expression of an encoding nucleic acid by ahuman cell expression system. This results in glycosylation of theexpressed spike receptor binding domain (S1RBD) protein at least at theN343 N-glycosylation site thereof, as shown in FIGS. 8 and 9, whichsurprisingly and significantly increases the affinity of the S1RBD forACE2. Use of the glycosylated form of the S1RBD, therefore, provides asignificant increase in the sensitivity of the assay compared to otherknown assays, increasing the ability of the assay to detect much smalleramounts or concentrations of the viral protein or its sensitivity toinhibitors then previously reported, and makes possible a more economicuse of reagents. Significantly, the glycosylated S1RBD polypeptide moreclosely resembles the glycosylated state of intact viral particlesproduced from an infected cell rather than does an unglycosylated S1RBDfragment that has been prepared by deglycosylation of a mammaliancell-produced S1RBD or by a bacterial expression system used for theirmanufacture, as shown in the data of FIGS. 4-11.

The present disclosure encompasses embodiments of the assay where theS1RBD protein fragment, when not immobilized on the surface and,therefore, intended to bind to immobilized ACE2 polypeptide includes atag conjugated thereto. Advantageously, this tag can be, but is notlimited to, an Fc portion of an immunoglobulin, most advantageously anIgG Fc region. This Fc tag can then be targeted by an anti-tag, such asan anti-immunoglobulin G (IgG) Fc-specific antibody) that has adetectable label attached thereto.

In one embodiment of the assay of the disclosure, the wells of amicrotiter plate are coated with a polypeptide derived from a mammalian,and most advantageously a human, ACE2 protein. An engineered recombinantmammalian cell-expressed S1RBD-derived polypeptide is then added to thecoated wells and then incubated for a time sufficient to allow bindingof the S1RBD fragment to the surface immobilized ACE2 fragment.

One fragment of the SARS-CoV-2 Spike (S) protein most advantageous foruse in the assays of the disclosure consists of amino acid residuesR319-R514 of the S1 region of the SARS-CoV-2 spike protein. Expressionof the S1RBD polypeptide from the mammalian cell expression systemresults in the expressed product being glycosylated. As shown in FIGS.4-11 it has now been found that this glycosylation thereby significantlyincreasing the affinity of the S1RBD for the ACE2 polypeptide, or afragment thereof.

Unbound S1RBD polypeptide is removed with washing, and a horse radishperoxidase (HRP)-conjugated anti-immunoglobulin G (IgG) antibodyspecific for the immunoglobulin Fc region can then be applied to thewells together with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate. TheHRP-conjugated anti-IgG antibody will bind specifically to the S1RBDpolypeptide bound to the surface-immobilized ACE2 polypeptide, reactwith the TMB solution, and produce a blue color, the intensity of whichis proportional to the amount of bound S1RBD. The HRP-TMB reaction ishalted with the addition of a Stop Solution, resulting in ablue-to-yellow color change. The intensity of the yellow color is thenmeasured at 450 nm.

This method of the disclosure may be advantageously employed to detect,or measure the amount of, the inhibition of the interaction betweenS1RBD and ACE2 by a compound suspected of being an inhibitor. To detector identify a potential inhibitor, a first assay is performed in theabsence of the compound suspected of being an inhibitor. A secondparallel test is performed simultaneously with the first test wherein anamount of the compound suspected of being the inhibitor is added to thesecond test with the S1 RBD polypeptide. A reduction in the intensity ofthe final yellow color of the second assay compared to the final colorin the first assay indicates that the compound inhibits the S1RBD/ACE2binding while the degree of the reduction can indicate the strength ofthe inhibition. Accordingly, the assays of the disclosure can be usefulto identify negative effectors of the binding of S1RBD to ACE2 that maybe useful in a therapeutic or prophylactic treatment for a SARS-CoV-2(Covid-19) virus infection, or even for use against related coronavirusinfections. Potential inhibitors can be, but are not limited to, smallmolecules (as shown in FIGS. 13 and 14) and antibodies (as shown inFIGS. 15-19),

This method is also useful for the detection of the intact spike proteinin a biological sample suspected of, for example, containing intactSARS-CoV-2 (Covid-19) virus or the surface antigens thereof. Compared toavailable means of detecting the Covid-19 spike protein in subjectssuspected of being infected with the virus, the assays of the presentdisclosure are more significantly more sensitive.

In second embodiment of the assay of the disclosure, the wells of amicrotiter plate are coated with mammalian cell-expressed S1 RBDpolypeptide. A fragment of a mammalian ACE2 protein (most advantageouslya human ACE2-derived polypeptide) is then added to the wells. UnboundACE2 polypeptide is removed with washing, and an anti-ACE2-specific IgGantibody is then applied to the wells. A horse radish peroxidase-labeled(for example) anti-IgG antibody is the added to the wells in thepresence of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate. Theanti-ACE2 antibody first binds to the ACE polypeptide bound to thesurface-immobilized S1RBD and then binds the labeled anti-IgG antibodyto react with the TMB solution, producing a blue color that isproportional to the amount of bound ACE2. The HRP-TMB reaction is haltedwith the addition of the Stop Solution, resulting in a blue-to-yellowcolor change. The intensity of the yellow color is then measured at 450nm.

In some embodiments, the ACE2-derived polypeptide can have a tagattached thereto, such as an immunoglobulin Fc tag. In the methods ofsuch an embodiment, the labeled secondary antibody can be an anti-tagantibody specifically binding to the tag of the ACE2 bound to theimmobile S1RBD.

The methods of the disclosure may also be readily adapted for use inlateral flow tests to provide a rapid method to detect an antiviralantibody or spike antigen in a sample such as a blood sample, nasal orsinus mucus, and the like. Such tests are less invasive, cheaper, andoffer significantly more rapid results than is provided by the“gold-standard” PCR test (detecting whole virus presence). Mostpreferably, the lateral flow device has immobilized ACE2 polypeptide adthe flow then encounters the S1RBD-tag and HRP-anti-tag antibody.

The second variant of the method of the disclosure may also beadvantageously employed to detect, or measure the amount of, theinhibition of the interaction between S1RBD and ACE2 by a compoundsuspected of being an inhibitor. In this case a first assay is performedin the absence of the compound suspected of being an inhibitor. A secondtest is performed simultaneously with the first test wherein an amountof the compound suspected of being the inhibitor is added to the secondtest with the ACE2 polypeptide. A reduction in the intensity of thefinal yellow color indicates that the compound is an inhibitor of theS1RBD/ACE2 binding and the degree of the reduction can indicate themagnitude of the inhibition.

The orientations of the two interacting components of the assay systemof the disclosure can equally be used to detect inhibitors of theS1RBD/ACE2 interaction. However, the orientation where the ACE2polypeptide is immobilized to the surface may also be used for thedetection of virus or free spike protein in a sample added to the wellwith the S1RBD reagent as well as useful for the detection of inhibitorsof S1RBD/ACE2 complexing. This embodiment provides a rapid sensitive andselective assay for the detection of intact SARS-CoV-2 (Covid-19) virusparticles in a biological sample from a subject suspected of having aninfection of the SARS-CoV-2 (Covid-19) virus.

The COVID-19 Spike-ACE2 binding assay kit of the disclosure providesmaterials and instructions for the rapid, simple, and sensitive methodof the disclosure to characterize the binding affinity of the S1RBD-ACE2complex in the presence of potential inhibitors. The in vitroenzyme-linked immunosorbent assay can measure numerous reagents andconditions simultaneously. For example, this kit can be used forscreening inhibitor activity and drugs, vaccine development, and testingpotential therapeutic antibodies.

One aspect of the disclosure, therefore, encompasses embodiments of amethod of detecting binding between the spike-receptor binding domain(S1RBD) of the SARS-CoV-2 (Covid-19) virus and angiotensin-convertingenzyme 2 (ACE2), the method comprising the steps: (a) contacting aglycosylated polypeptide derived from a spike-receptor binding domain(S1RBD) of the SARS-CoV-2 (Covid-19) virus spike (S) protein with apolypeptide derived from a mammalian ACE2, wherein the S1RBD polypeptideor the ACE2-derived polypeptide is bound to the surfaces of wells of amicrotiter plate, wherein the S1RBD polypeptide is a recombinantglycosylated polypeptide expressed from a mammalian cell expressionsystem; (b) washing the wells of unbound polypeptides; (c) either: (i)when the surface bound polypeptide is the ACE2 polypeptide, contactingthe surface bound ACE2 polypeptide with the glycosylated S1RBDpolypeptide, wherein the S1RBD polypeptide further comprises a tagconjugated thereto; or, (ii) when the surface bound polypeptide is theglycosylated S1RBD polypeptide, contacting the surface bound polypeptidewith the ACE2 polypeptide, and then incubating the wells for a periodthat allows the polypeptide bound to the well surfaces to form a complexwith to the polypeptide delivered thereto; (d) washing the wells ofunbound polypeptides; (e) delivering to the wells from step (c)(i) adetectably labeled anti-tag-specific antibody or delivering to the wellsfrom step (c)(ii) a detectably labeled anti-ACE2-specific antibody; (f)incubating the wells for a period to allow the antibody deliveredthereto to bind to the complex formed in either step (c)(i) or (c)(ii);and (g) detecting the label on an antibody bound to the compleximmobilized on the microtiter plate, thereby detecting binding of theS1RBD to the ACE2.

In some embodiments of this aspect of the invention, the polypeptidebound to wells of a microtiter plate can be an ACE2 polypeptide and iscomplexed in step (c)(i) to S1RBD-tag polypeptide delivered to thewells. This method can further comprise the steps: (g) repeating theassay steps (a)-(f) in the presence of a biological sample suspected ofcomprising SARS-CoV-2 (Covid-19) virus, wherein in step (a) the sampleis added to the wells of the microtiter plate; and (h) measuring thedifference between the signal from the detectable label in the absenceand presence of the sample suspected of comprising SARS-CoV-2 (Covid-19)virus, wherein a reduction in the intensity of the signal generated inthe presence of the compound indicates that the sample comprisesSARS-CoV-2 (Covid-19) virus.

In some embodiments of this aspect of the invention the polypeptidebound to wells of a microtiter plate is a glycosylated S1RBD polypeptideexpressed from a mammalian cell expression system and is complexed instep (c)(ii) to the ACE2 polypeptide delivered to the wells.

In some embodiments of this aspect of the invention the tag conjugatedto the S1RBD polypeptide can be an immunoglobulin G (IgG) Fc region andthe anti-tag-specific antibody can be an anti-IgG Fc-specific antibody.

In some embodiments of this aspect of the invention the S1RBDpolypeptide can comprise the amino acid sequence SEQ ID NO: 1.

In some embodiments of this aspect of the invention the S1RBDpolypeptide can comprise the amino acid sequence SEQ ID NO: 1 and isglycosylated at least at the N343 N-glycosylation site thereof.

In some embodiments of this aspect of the invention the label can behorse radish peroxidase (HRP).

In some embodiments of this aspect of the invention the method canfurther comprise the steps: (g) repeating the assay steps (a)-(f) in thepresence of a compound suspected of being an inhibitor of the binding ofthe S1RBD polypeptide to the ACE2 polypeptide or a biological samplesuspected of containing SARS-CoV-2 (Covid-19) virus or an antibodythereto, wherein in steps (c)(i) and (c)(ii) the compound is added tothe wells of the microtiter plate; and (h) measuring the differencebetween a signal from the detectable label in the absence and presenceof the compound suspected of being an inhibitor of the binding of theS1RBD polypeptide to the ACE2 polypeptide, wherein a reduction in theintensity of the signal generated in the presence of the compoundindicates that the compound is an inhibitor of the S1RBD/ACE2 bindingand the degree of the reduction can indicate the magnitude of theinhibition.

In some embodiments of this aspect of the invention the compoundsuspected of being an inhibitor of the binding of the S1RBD polypeptideto the ACE2 polypeptide can be a small molecule, an antibody, or apeptide.

In some embodiments of this aspect of the invention, the antibody can bea monoclonal antibody or in a biological sample isolated from a patientsuspected of having generated anti-SARS-CoV-2 (Covid-19) virusantibodies.

Another aspect of the disclosure encompasses embodiments of a kitcomprising; at least one microtiter plate comprising a plurality ofwells, wherein said wells are coated with an Angiotensin ConvertingEnzyme 2 (ACE2) extracellular domain-derived polypeptide; a plurality ofvessels, wherein said vessels can contain a wash buffer, an assaydiluent, a purified glycosylated SARS-CoV-2 (Covid-19) spike protein RBDregion (S1RBD)-derived polypeptide, wherein the S1RBD polypeptide isobtained by expression from a mammalian cell, and wherein the S1RBDprotein has an immunoglobulin Fc tag conjugated thereto; a horse radishperoxidase-conjugated anti-immunoglobulin G (IgG) Fc-region antibody, aTMB One-Step Substrate Reagent comprising 3,3′,5,5′-tetramethylbenzidine(TMB) in a buffer; and a reaction stop solution comprising about 0.2Msulfuric acid; and instructions for the use of the kit to assay thebinding of the glycosylated S1RBD polypeptide to a domain of ACE2 in theabsence and presence of a compound or a biological sample suspected ofinhibiting said binding.

In some embodiments of the kit of the disclosure, the kit can compriseat least one microtiter plate comprising a plurality of wells, whereinsaid wells are coated with a glycosylated SARS-CoV-2 (Covid-19) spikeprotein RBD region (S1RBD)-derived polypeptide, wherein the S1RBDpolypeptide is obtained by expression from a mammalian cell, a pluralityof vessels, wherein said vessels can contain a wash buffer, an assaydiluent, a purified extracellular domain of a recombinant ACE2polypeptide, a horse radish peroxidase-conjugated anti-ACE2 antibody, aTMB One-Step Substrate Reagent comprising 3,3′,5,5′-tetramethylbenzidine(TMB) in a buffer, and a reaction stop solution comprising about 0.2Msulfuric acid; and instruction for the use of the kit to assay thebinding of the glycosylated S1RBD polypeptide to a domain of ACE2 in theabsence and presence of a compound or biological sample suspected ofinhibiting said binding.

While embodiments of the present disclosure are described in connectionwith the Examples and the corresponding text and figures, there is nointent to limit the disclosure to the embodiments in these descriptions.On the contrary, the intent is to cover all alternatives, modifications,and equivalents included within the spirit and scope of embodiments ofthe present disclosure.

EXAMPLES Example 1

Kit Material Provided

ACE2 Microplate (Item A): 96 wells (12 strips×8 wells) coated withrecombinant ACE2 extracellular domain.

Wash Buffer Concentrate (20×: Potassium Chloride, 0.4%; Sodium Chloride,16%, Potassium Dihydrogen Phosphate; 0.4%, Disodium Phosphate; Tween 20,1.0%) (Item B): 25 ml of 20×concentrated solution.

Assay Diluent (Item E2): 15 ml of 5×concentrated buffer (5×PhosphateBuffered Saline (PBS); 7.5 wt % Bovine Serum Albumin (BSA); 0.15%5-Bromo-5-nitro-1,3-dioxane (BND); 1.25% Tween-20) for diluting testingreagent, S1RBD-tag protein (Item F), detection antibody (Item C) andHRP-conjugated anti-Fc region antibody concentrate (Item D).

S1RBD-tag protein (Item F): 2 vials of purified human recombinantglycosylated S1RBD-tag protein (1 vial is enough to assay halfmicroplate)

HRP-conjugated anti-Fc region antibody (Item D-2), 25 μl of1000×concentrated HRP-conjugated anti-goat IgG.

TMB One-Step Substrate Reagent (Item H): 12 ml of3,3′,5,5′-tetramethylbenzidine (TMB) in buffered solution.

Stop Solution (Item I): 8 ml of 0.2 M sulfuric acid.

Storage

Upon receipt, the kit should be stored at −20° C. or below and usedwithin 6 months from the date of shipment. After initial use, WashBuffer Concentrate (Item B), Assay Diluent (Item E2), TMB One-StepSubstrate Reagent (Item H), Stop Solution (Item I) should be stored at4° C. to avoid repeated freeze-thaw cycles. Return unused wells to thepouch containing a desiccant pack, reseal along entire edge and store at−20° C. Item F should be stored at −80° C. Item C and Item D store at2-8° C. for up to one month (store at −20° C. for up to 6 months, avoidrepeated freeze-thaw cycles).

Additional Materials Required

Microplate reader capable of measuring absorbance at 450 nm.

Shaker.

Precision pipettes to deliver 2 □l to 1 ml volumes.

Adjustable 1-25 ml pipettes for reagent preparation.

100 ml and 1 liter graduated cylinders.

Distilled or deionized water.

Tubes to prepare sample dilutions.

Example 2

Sample Preparation

Mix testing reagent (e.g., potential inhibitor such as, but not limitedto, a small molecule or an antibody) with S1RBD-tag protein concentrate(see Part VI, 4), then dilute the mixture with 1×Assay Diluent dilutedto make a 1×51RBD-tag protein working concentration. Each sample shouldcontain the same 1×51RBD-tag protein concentration.

All samples be run in at least duplicate. For the initial experiment, aserial dilution (e.g., 5-fold to 5000-fold) can be performed todetermine the optimal amount of test reagent to use. Example: To testcompound A's ability to inhibit S1RBD-tag protein-ACE2 binding, dilutethe 100 mM stock solution to create a dilution series of 20 mM, 2 mM,0.2 mM, 0.02 mM, 0.002 mM and 0 mM in six separate tubes.

Pipette 225 μl of 1×51RBD-tag protein working solution into each tube,except the 20 mM (leave this one empty). Pipette 50 μl of compound Astock, 2.5 μl of S1RBD-tag protein concentrate and 197.5 ul 1×AssayDiluent into the tube labeled 20 mM. Mix thoroughly. Pipette 25 μl ofthe 20 mM compound A sample into the tube labeled 2 mM. Mix thoroughly.Repeat this step with each successive concentration. For each dilution,use 225 μl of 1×S1RBD-tag protein working solution and 25 μl of theprior concentration until the final concentration is reached. Mix eachtube thoroughly before the next transfer.

Example 3

Reagent Preparation

1. Bring all reagents and samples to room temperature (18-25° C.) beforeuse.

2. 5×Assay Diluent (Item E2) should be diluted 5-fold with deionized ordistilled water before use.

3. If the Wash Concentrate (20×) (Item B) contains visible crystals,warm to room temperature and mix gently until dissolved. Dilute 20 ml ofWash Buffer Concentrate into deionized or distilled water to yield 400ml of 1×Wash Buffer.

4, Briefly spin the S1RBD-tag protein (Item F) before use. Add 100 μl of1×Assay Diluent into the vial to prepare an S1RBD-tag proteinconcentrate. Pipette up and down to mix gently (the concentrate can bestored at 4° C. for 1-2 days or at −80° C. for one month). The S1RBD-tagprotein working solution should be diluted 100-fold with 1×Assay Diluentand used in sample preparation.

5. Briefly spin the HRP-conjugated anti-Fc region antibody (Item D-1)before use. HRP-conjugated anti-Fc region antibody concentrate should bediluted 1000-fold with 1×Assay Diluent.

EXAMPLE: Briefly Spin the Vial to Collect Contents to the Bottom. Add 5μl of HRP-Conjugated anti-Fc region antibody concentrate into a tubewith 5 mL 1×Assay Diluent, then pipette up and down to mix gently toprepare a 1000-fold diluted HRP-conjugated anti-Fc region antibodysolution. Mix well.

Example 4

Assay Procedure:

1. Bring all reagents to room temperature (18-25° C.) before use.

2. Add 100 μl of each sample (S1RBD protein with or without a possibleS1RBD-ACE2 binding inhibitor) into an appropriate well.

Note: It is recommended that all samples should be run in at leastduplicate.

3. Cover well with plate holder and incubate for 2.5 hours at roomtemperature or overnight at 4° C. with shaking.

4. Discard the solution and wash 4 times with 1×Wash Solution. Wash byfilling each well with 1×Wash Buffer (300 μl) using a multi-channelpipette or autowasher. Complete removal of liquid at each step isessential to good performance. After the last wash, remove any remaining1×Wash Buffer by aspirating or decanting. Invert the plate and blot itagainst clean papertowels.

5. Add 100 μl of prepared 1×HRP-conjugated anti-Fc region antibody (seeReagent Preparation Step 6) to each well. Incubate for 1 hour at roomtemperature with shaking.

6. Discard the solution. Repeat the wash as described in Step 3.

7. Add 100 μl of TMB One-Step Substrate Reagent (Item H) to each well.Incubate for 30 minutes at room temperature in the dark with shaking.

8. Add 50 μl of Stop Solution (Item I) to each well. Read at 450 nmimmediately.

Example 5

Kit Material Provided

COVID19 S-protein Microplate (Item A): 96 wells (12 strips×8 wells)coated with recombinant mammalian cell generated (glycosylated) COVID19S-protein RBD domain.

Wash Buffer Concentrate (20×: Potassium Chloride, 0.4%; Sodium Chloride,16%, Potassium Dihydrogen Phosphate; 0.4%, Disodium Phosphate; Tween 20,1.0%) (Item B): 25 ml of 20×concentrated solution.

Assay Diluent (Item E2): 15 ml of 5×concentrated buffer (5×PhosphateBuffered Saline (PBS); 7.5 wt % Bovine Serum Albumin (BSA); 0.15%5-Bromo-5-nitro-1,3-dioxane (BND); 1.25% Tween-20) for diluting testingreagent, ACE2 protein (Item F), detection antibody (Item C) andHRP-conjugated IgG concentrate (Item D).

ACE2 protein (Item F): 2 vials of purified human recombinant ACE2protein (1 vial is enough to assay half microplate)

ACE2 Detection Antibody (Item C-1): 2 vials of goat anti-ACE2 (1 vial isenough to assay half microplate).

HRP-conjugated anti-goat IgG (Item D-1), 25 μl of 1000×concentratedHRP-conjugated anti-goat IgG.

TMB One-Step Substrate Reagent (Item H): 12 ml of3,3′,5,5′-tetramethylbenzidine (TMB) in buffered solution.

Stop Solution (Item I): 8 ml of 0.2 M sulfuric acid.

Storage

Upon receipt, the kit should be stored at −20° C. or below and usedwithin 6 months from the date of shipment. After initial use, WashBuffer Concentrate (Item B), Assay Diluent (Item E2), TMB One-StepSubstrate Reagent (Item H), Stop Solution (Item I) should be stored at4° C. to avoid repeated freeze-thaw cycles. Return unused wells to thepouch containing a desiccant pack, reseal along entire edge and store at−20° C. Item F should be stored at −80° C. Item C and Item D store at2-8° C. for up to one month (store at −20° C. for up to 6 months, avoidrepeated freeze-thaw cycles).

Additional Materials Required

Microplate reader capable of measuring absorbance at 450 nm.

Shaker.

Precision pipettes to deliver 2 □l to 1 ml volumes.

Adjustable 1-25 ml pipettes for reagent preparation.

100 ml and 1 liter graduated cylinders.

Distilled or deionized water.

Tubes to prepare sample dilutions.

Example 6

Sample Preparation

Mix testing reagent (e.g., potential inhibitor such as, but not limitedto, a small molecule or an antibody) with ACE2 protein concentrate (seePart VI, 4), then dilute the mixture with 1×Assay Diluent dilute to makea 1×ACE2 protein working concentration. Each sample should contain thesame 1×ACE2 protein concentration.

All samples be run in at least duplicate. For the initial experiment, aserial dilution (e.g., 5-fold to 5000-fold) can be performed todetermine the optimal amount of test reagent to use. Example: To testcompound A's ability to inhibit Spike-ACE2 binding, dilute the 100 mMstock solution to create a dilution series of 20 mM, 2 mM, 0.2 mM, 0.02mM, 0.002 mM and 0 mM in six separate tubes.

Pipette 225 μl of 1×ACE2 protein working solution into each tube, exceptthe 20 mM (leave this one empty). Pipette 50 μl of compound A stock, 2.5μl of ACE2 protein concentrate and 197.5 ul 1×Assay Diluent into thetube labeled 20 mM. Mix thoroughly. Pipette 25 μl of the 20 mM compoundA sample into the tube labeled 2 mM. Mix thoroughly. Repeat this stepwith each successive concentration. For each dilution, use 225 μl of1×ACE2 protein working solution and 25 μl of the prior concentrationuntil the final concentration is reached. Mix each tube thoroughlybefore the next transfer.

Example 7

Reagent Preparation

1. Bring all reagents and samples to room temperature (18-25° C.) beforeuse.

2. 5×Assay Diluent (Item E2) should be diluted 5-fold with deionized ordistilled water before use.

3. If the Wash Concentrate (20×) (Item B) contains visible crystals,warm to room temperature and mix gently until dissolved. Dilute 20 ml ofWash Buffer Concentrate into deionized or distilled water to yield 400ml of 1×Wash Buffer.

4, Briefly spin the ACE2 protein (Item F) before use. Add 100 μl of1×Assay Diluent into the vial to prepare an ACE2 protein concentrate.Pipette up and down to mix gently (the concentrate can be stored at 4°C. for 1-2 days or at −80° C. for one month). The ACE2 protein workingsolution should be diluted 100-fold with 1×Assay Diluent and used insample preparation.

5. Briefly spin the detection antibody (Item C-1) before use. Add 100 μlof 1×Assay Diluent into the vial to prepare a detection antibodyconcentrate. Pipette up and down to mix gently (the concentrate can bestored at 4° C. for 5 days or at −80° C. for one month). The goatanti-ACE2 antibody concentrate should be diluted 55-fold with 1×AssayDiluent and used in step 4 of the assay procedure.

6. Briefly spin the HRP-conjugated anti-goat IgG (Item D-1) before use.HRP-conjugated anti-goat IgG concentrate should be diluted 1000-foldwith 1×Assay Diluent. EXAMPLE: Briefly spin the vial to collect contentsto the bottom. Add 5 μl of HRP-conjugated anti-goat IgG concentrate intoa tube with 5 mL 1×Assay Diluent, then pipette up and down to mix gentlyto prepare a 1000-fold diluted HRP-conjugated anti-goat IgG solution.Mix well.

Example 8

Assay Procedure:

1. Bring all reagents to room temperature (18-25° C.) before use.

2. Add 100 μl of each sample into an appropriate well.

Note: It is recommended that all samples should be run in at leastduplicate.

3. Cover well with plate holder and incubate for 2.5 hours at roomtemperature or overnight at 4° C. with shaking.

4. Discard the solution and wash 4 times with 1×Wash Solution. Wash byfilling each well with 1×Wash Buffer (300 μl) using a multi-channelpipette or autowasher. Complete removal of liquid at each step isessential to good performance. After the last wash, remove any remaining1×Wash Buffer by aspirating or decanting. Invert the plate and blot itagainst clean papertowels.

5. Add 100 μl of prepared 1×Detection Antibody (Reagent Preparation step5) to each well. Incubate for 1 hour at room temperature with shaking.

Discard the solution. Repeat the wash as described in Step 3.

6. Add 100 μl of prepared 1×HRP-conjugated anti-goat IgG (see ReagentPreparation Step 6) to each well. Incubate for 1 hour at roomtemperature with shaking.

7. Discard the solution. Repeat the wash as described in Step 3.

8. Add 100 μl of TMB One-Step Substrate Reagent (Item H) to each well.Incubate for 30 minutes at room temperature in the dark with shaking.

9. Add 50 μl of Stop Solution (Item I) to each well. Read at 450 nmimmediately.

Example 9

In vitro binding of viral components to human ACE2: A high throughputscreening method to measure molecular binding between the SARS-CoV-2 Sprotein and the human ACE2 protein. Microtiter plates were coated witheither recombinant SARS-CoV-2 S protein 51 domain, S2 domain, ornucleocapsid (N) protein. Systematic incubation of these plates withrecombinant human ACE2 confirmed that ACE2 specifically bound to S1RBDand not to any of the other viral components tested (FIG. 10A).Furthermore, recombinant SARS-CoV-2 S1RBD generated by expression withinhuman HEK293T cultured cells bound to the ACE2 with significantlystronger affinity compared to recombinant 51 RBD generated by expressionwithin E. coli (FIG. 10B). These data indicate that eukaryotic-specificpost-translational modifications influences ACE2/S1RBD binding affinity.

Example 10

N-linked glycosylation of S1 RBD protein is required for binding toACE2: To determine the extent to which glycosylation influencesACE2/S1RBD binding affinity, S1RBD generated within HEK293T culturedcells was deglycosylated to remove both N and O-linked glycans. Whereasuntreated S1RBD bound to ACE2 in a dose-dependent manner,deglycosylation of 51 RBD abolished its binding to ACE2 (FIG. 6).Importantly, deglycosylation of ACE2 had a minimal effect on binding(FIG. 6). To further interrogate which glycan linkages function inACE2/S1 RBD binding, S1RBD was treated with deglycosylation enzymes thatspecifically target different glycosylation links. Of the enzymestested, only treatment with PNGase, which cleaves N-linkedoligosaccharides, significantly lowered ACE2/S1 RBD binding affinity(FIG. 7).

Example 11

Glycosylation of S1RBD at Asn343 is essential for interaction with ACE2:The SARS-CoV-2 S protein has 22 putative glycosites as determined by thepresence of the N-X-S/T, X≠P motif sequence (Zhang et al., (2020) Mol.Cell. Proteomics). Of these sites, N331 and N343 are located on the RBDand have been shown to be n-glycosylated (Zhang et al., (2020) Mol.Cell. Proteomics) and when mutated to glutamine were found todrastically reduce viral infectivity (Li et al., (2020) Cell 182:1284-1294). To determine whether these residues function in ACE2/S1RBDmolecular interaction during binding, three mutated recombinants of S1RBD were generated: N343Q, N331Q, and a N343Q/N331Q double mutation.Measurement of in vitro binding activity of these mutants to ACE2revealed that binding was abolished in both N343Q and N331Q/N343Q andwas significantly lower in the N331Q mutant version of S1 RBD (FIG. 7).

Example 12

Mutation of glycosylation sites significantly reduces pseudoviralinfectivity: To measure the influence of the N343 and N331 glycosites oninfectivity, a pseudovirus system was used that expresses the SARS-CoV-2S1 RBD on a viral particle surface, while a plasmid encoding forluciferase is contained inside the particle. When applied to culturedmammalian cells, the S protein binds the ACE2 receptor, the membranes ofthe viral particle and host cell fuse, releasing the plasmid into thecell where luciferase is expressed. In addition to the wild type versionof the S1RBD, pseudovirus was generated that expressed on the viralsurface mutated versions of S1RBD, namely N343Q, N331Q, and aN343Q/N331Q double mutation. Measurement of luciferase activity withininfected cells showed that the double glycosylation deletions at N331and N343 resulted in a substantial reduction in viral infectivity (>80%inhibition), whereas single deletion at N331Q caused modest with theinfectivity of N331Q (reduced by less than 20%) and N343Q by about 50%(FIG. 8).

These data demonstrate that double glycosylation mutations in S1RBD(N331Q and N343Q) significantly reduced infectivity, suggesting that thetwo glycosylation sites in the RBD region may participate in the bindingof the receptor or maintain the conformation of the RBD region.Expression of wild type spike and three Spike mutants was confirmed inthe VSV-Spike viral particles.

Example 13

Monoclonal anti-SARS-COV-2 S1RBD antibody inhibits SARS-COV-2-S-drivenentry: It was tested whether antibodies against the RBD could blockSARS-2-S driven entry. Mouse anti-S1RBD monoclonal antibodies werescreened for the neutralization activity using the pseudoviralneutralization test (PVNT) assay.

As shown in FIG. 9, monoclonal antibodies 807, 814, 844, and 845inhibited more than 50% of SARS-COV-2-S-driven entry at 2 μg/ml. MM57, acommercial neutralizing antibody (Sinobio) served as a positive control.

Example 14

COVID-19 patient sera exhibit a high level of neutralization activity(blockade of SARS-COV-2-S driven entry): To determine the applicabilityof the neutralization test for measuring neutralizing activity ofCOVID-19 patient sera, 15 sera obtained from convalescent COVID-19patients were collected and tested using the PVNT assay. The resultsindicated that all of them inhibited SARS-COV-2-S-, but not VSV-G-,driven entry in a concentration-dependent manner (FIG. 19). Noinhibition was observed when the serum from health person used as anormal control (FIG. 19).

Example 15

Zafirlukast, a small molecule, neutralizes SARS-COV-2 Spike-drivenentry: Small molecule inhibitors were tested using the PVNT.Zafirlukast, a small molecule inhibited SARS-COV-2-S-but notVSV-G-driven entry in a concentration-dependent manner (FIG. 13). Noinhibition was observed when cefoperazone and camostat or DMSO (FIG.13). A Western blot analysis confirmed the expression of spike protein(approximately 165 kDa band) in the VSV-spike pseudoviral particlesprobed with a mouse anti SARS-COV-2 S1RBD monoclonal antibody(130-10864). No signal was detected in the VSV-G pseudoviral particles.

What is claimed:
 1. A method of detecting an inhibitor of bindingbetween the spike-receptor binding domain (S1RBD) of the SARS-CoV-2(Covid-19) virus and angiotensin-converting enzyme 2 (ACE2), the methodcomprising the steps: (a) contacting a glycosylated fragment of thespike-receptor binding domain (S1RBD) of the SARS-CoV-2 (Covid-19) virusspike (S) protein with a polypeptide fragment from a mammalian ACE2protein, wherein the S1RBD polypeptide fragment is bound to the wells ofa microtiter plate, is a recombinant glycosylated polypeptide expressedfrom a mammalian cell expression system, and has the amino acid sequenceSEQ ID NO: 1, and the ACE2 fragment has the amino acid sequence SEQ IDNO: 2; (b) contacting the well-bound glycosylated S1RBD fragment withthe ACE2 fragment, and then incubating the wells for a period thatallows the well-bound glycosylated S1RBD fragment to form a well-boundcomplex with the ACE2 fragment delivered thereto; (c) washing the wellsof unbound polypeptides; (d) delivering to the wells from step (c) adetectably labeled anti-ACE2-specific antibody; (e) incubating the wellsfor a period to allow the anti-ACE2-specific antibody to bind to thewell-bound complex formed in step (b); and (f) washing the wells ofunbound polypeptides; (g) detecting a signal generated by the label onthe anti-ACE2-specific antibody bound to the well-bound complex, therebydetecting binding of the S1RBD fragment to the ACE2 fragment; (h)repeating steps (a)-(g), wherein in step (a) a suspected inhibitor ofS1RBD/ACE2 binding is added to the wells of the microtiter plate,wherein the suspected inhibitor is either a small molecule, or apeptide; and (i) measuring the difference between the signals from thedetectable label in the absence and presence of the suspected S1RBD/ACE2binding inhibitor, wherein lower signal intensity generated in thepresence of the suspected inhibitor compared to the signal intensity inthe absence of the suspected inhibitor indicates that the suspectedinhibitor is an inhibitor or comprises an inhibitor of S1RBD/ACE2binding and the degree of the signal intensity reduction furtherindicates the magnitude of the inhibition.
 2. The method of claim 1,wherein the label is horse radish peroxidase (HRP).
 3. The method ofclaim 1, wherein the ACE2 fragment has the amino acid sequence SEQ IDNO: 2.